It is important that an Expressed Sequence Tag (EST) database produced for a model organism represents rnRNA transcripts from all possible tissues. We propose to use a transgenic approach to help identify cell lineage-specific ESTs from developing zebrafish embryos. The focus of this proposal will be the identification of ESTs from embryonic neuronal cells. To this end, we have generated transgenic zebrafish that express the green fluorescent protein (GFP) reporter gene specifically in embryonic neuronal cells. GFP-positive neuronal cells will be purified from living embryos using fluorescence activated cell sorting (FACS). RNA isolated from the FACS-purified cells will then be used to construct cDNA libraries. These libraries will be submitted to the NIH-sponsored Washington University EST Center for large scale DNA sequencing analysis and sequence information will be entered into public database directly from the EST center. We will search the database with our EST sequences to "weed out" those genes already identified. EST sequences whose function is not yet identified will be considered to be "novel" and will be used to analyze expression patterns and chromosomal locations. Genes identified in these experiments, coupled with the knowledge generated concerning their expression patterns and chromosomal assignment, should serve as excellent candidate genes for the zebrafish embryonic mutations affecting neuronal development.